Tuberculosis (TB) remains the leading infectious cause of global mortality. The emergence of drug resistant TB and a deadly synergy with HIV seen in recent years highlight the need for a better understanding of TB transmission and pathogenesis. The long term objectives of our project are: 1) to identify underlying genetic factors of Mycobacterium tuberculosis important to the pathogenesis and the epidemiology of TB and 2) to provide targets for the development of more efficient vaccine and therapeutical agents for TB prevention and control. Our hypothesis for the present project is that survival of successful bacterial pathogens not only depends on their ability to alter global patterns of gene expression in response to the changing environments during infection, but also depends on the presence or absence of certain genes in the bacteria that play an important role in virulence, and that loss or gain of such genes in clinical strains of M. tuberculosis is associated with their infectivity and pathogenicity. Our specific aims and the related experimental strategies are: 1) to determine genomic variation among M. tuberculosis strains associated with different epidemiological and clinical characteristics by genomic subtraction and to identify DNA unique to epidemiologically and clinically successful strains of M. tuberculosis in comparison with epidemiologically and clinically less successful strains, and with already sequenced strains of M. tuberculosis, H37Rv and CDC 1551; 2) to assess the potential importance of the fragments identified by DNA subtraction to TB transmission and pathogenesis by screening for the presence or absence of the fragments in approximately 700 clinical isolates collected during a five year population-based molecular epidemiological study conducted in Arkansas; and the relative distribution of these subtraction products will be determined by epidemiological and clinical co-variates; and 3) to identify and to characterize genes contained in the subtraction products which are associated with TB transmission and pathogenesis, the flanking regions of the fragments identified by subtraction will be cloned for sequencing. By combining epidemiological information with molecular genetics, we are conducting a focused search for genes associated with transmission and pathogenesis.